Endogenous sex steroids in

premenopausal women and risk of breast

cancer: the ORDET cohort
Introduction
Sex steroids regulate cell proliferation and play a major role in breast cancer
promotion [1,2]. Particularly androgens and estrogens have been associated with breast
cancer risk in postmenopausal women [3]. Few previous studies have evaluated the
association between circulating sex steroids and breast cancer risk in premenopausal women,
and in general, they [4-16] do suggest a consistent association with breast cancer risk only for
total and bioavailable testosterone, whereas the role of estradiol and progesterone has been
less clear and inconsistent, thus far [1]. These studies, however, tended to be limited by
intraindividual hormone variability due to the ovarian cycle and by technical variability in
hormone assays. We sought to clarify further the strength of the association between
prediagnostic sex steroid hormones and breast cancer incidence in premenopausal women.
The prospective nature of our study, careful control of hormone variability through both a
specifically developed study design and highly standardized protocols for specimen
collection and storage, availability of stored biologic samples, and detailed information on
lifestyle, reproductive history, and standardized anthropometric measurements, together with
a median cohort follow-up of 20 years and the identification of all breast cancer cases assured
by the local Varese Cancer Registry, render the Hormones and Diet in the Etiology of Breast
Cancer Risk (ORDET) study an ideal setting for better clarification of hormonal risk factors
in relation to breast cancer development [17-19].
This current analysis is based on a substantial update of a previous analysis [12],
which used only roughly one fifth of all women with breast cancer that constitute the current
sample. We evaluated associations between mid-luteal total and free testosterone (FT),
estradiol, progesterone, and sex hormone-binding globulin (SHBG) and premenopausal
breast cancer risk. We also considered in the analysis follicle-stimulating hormone (FSH) and
luteinizing hormone (LH).

Methods
The ORDET cohort was established in northern Italy between June 1987 and June
1992, when 10,786 healthy women, ages 35 to 69 years, were enrolled [20,21]. They were all
residents of the Varese province, an area covered by the Varese Cancer Registry [22], who
had heard about the study through the media, at public meetings, and at breast cancer early-
detection centers, and who volunteered to participate. At recruitment, a number of baseline
characteristics, including demographics and dietary intake, were queried from each
participant via questionnaire. Direct measurements of several anthropometric variables,
including height and weight, were conducted, and blood and urine specimens were collected.
Because of the focus of the study on endogenous hormones and their relation with breast
cancer risk, stringent inclusion criteria were established, and highly standardized conditions
on collecting biologic samples were applied. Women were excluded if they reported a
bilateral oophorectomy, were currently breast feeding or pregnant, used oral contraceptives or
hormone-replacement therapy in the last 3 months, were affected by chronic or acute liver
disease, or reported a history of cancer (with the exception of nonmelanoma skin cancer).
Information on cancer outcomes, which was available from the local cancer registry
(Varese Cancer Registry), was linked to the ORDET cohort to identify incident breast cancer
cases up to December 2003. The Varese Cancer Registry is of high quality: <2% of breast
cancer cases are known to the registry by death certificate only, and the histology and
cytology of 96.3% of all cases has been confirmed through pathology reports [20,23]. The
ORDET file was also linked to the Varese residents’ files to check participants’ vital status.
Participants were censored at the time of cancer diagnosis, death, or loss to follow-up,
whichever came first (median follow-up time, 15.4 years). Written consent was obtained
from all study participants, and the study was approved by the Ethical Review Board of the
National Cancer Institute of Milan (Italy).

Assessment of menopausal status and selection of study subjects

Case subjects were women who developed breast cancer after their recruitment into
the ORDET cohort but before the end of the study period (December 31, 2003). For each case
subject with breast cancer, up to four control subjects were randomly chosen from
appropriate risk sets consisting of all cohort members who satisfied the matching criteria and
were alive and free of cancer at the time of diagnosis of the index case. Matching
characteristics were age (±3 years) at enrollment and date of recruitment (±180 days). Once
each breast cancer case was matched with her controls, to reduce the interbatch technical
variability, we assessed their hormonal levels within the same laboratory batch. An incidence
density-sampling protocol for control selection was used, such that controls could include a
subject who became a case later, whereas each control subject could also be sampled more
than once.
After exclusion of women with a history of cancer and women who, immediately after
baseline, were lost to follow-up (observed time = 0), 10,633 participants remained to form the
base population of ORDET. For this study, we further restricted the ORDET cohort to its
6,667 premenopausal participants. Women were classified as premenopausal, at recruitment,
if they had at least one menstruation in the 6 months before recruitment. All breast cancer
cases were premenopausal at blood draw. To define premenopause more stringently, only
women with FSH levels <30 μl/ml were initially included (205 cases and 767 controls). Of
these 205 cases, 20 had in situ breast cancer. We conducted separated analyses including and
excluding the 20 breast cancers in situ. The results were similar, and we decided to describe
the study results with the inclusion of the breast cancer in situ.
In addition, to control by study design the biologic variability due to irregular ovarian
cycles, we considered only cases and control women who reported menstrual cycles at fairly
regular intervals throughout their life (126 breast cancer cases and 474 control subjects) and
excluded women with marked menstrual irregularities (that is, women with more than one
period missing in a 6-month period: 79 breast cancer cases and matched 293 control subjects)
and women who had the menstrual cycle at blood collection longer than 45 days (13 breast
cancer cases and 49 matched controls). These restrictions left nine cases without matched
controls and 200 controls without matched cases, hence 104 case women and 225 controls
were included in this study.

Blood collection
A detailed description of blood-collection methods was provided previously [12,17].
In brief, blood samples were collected after overnight fasting between 7:30 AM and 9:00 AM
from each woman. They were timed to be collected between days 20 and 24 of their
menstrual cycle (that is, during the midluteal phase). For further verification of their luteal
phase during blood collection, women were given a postcard to report the date of the
subsequent bleeding after the blood drawing. Of 6,667 women who had at least one
menstruation in the 12 months before recruitment, 6,030 (90.4%) returned their postcard with
information on the date of the menses subsequent to the blood collection. Blood samples
were stored at -80°C until the present hormone determinations.

Laboratory methods
Stability and reliability of the ORDET collection method for sex steroids in
premenopausal women were previously described [12]. Blood samples from breast cancer
cases and related controls were handled identically and assayed together on the same day and
in the same run.
All samples were taken out of the freezer simultaneously and sent to the laboratory in
the same parcel on dry ice. Laboratory personnel were blinded to case-control status. Control
of analytic error was based on the inclusion of two standard samples. All samples were
assayed in duplicate, by using commercially available kits following the manufacturer’s
instructions. Plasma sex steroid measurements (testosterone, free testosterone, SHBG, and
estradiol) were conducted by Centro Medico Diagnostico Emilia (Bologna, Italy). For
testosterone and free testosterone, we used Coat-A-Count procedure, a solid-phase
radioimmunoassay (Diagnostic Products Corporation, Los Angeles, CA, USA); for SHGB,
IMMUNOLITE 1000 Analyzer, a solid-phase, chemiluminescent immunometric assay
(Diagnostic Products); and for estradiol, Orion Diagnostica SPECTRIA Estradiol Sensitive
RIA test, a coated-tube radioimmunoassay (Orion Diagnostica Oy, Espoo, Finland). Quality
control was done at three concentrations for SHBG and total and free testosterone and at four
concentrations for total estradiol. In each batch, quality-control samples were evaluated in
quadruplicate. Within-batch quality control coefficients of variation for high and low
concentrations were 5.9% and 14.0% for estradiol; 5.8% and 10.6% for total testosterone;
7.0% and 9.6% for free testosterone; and 3.1% and 3.4% for SHBG. Average between-batch
coefficients of variation for high and low concentrations were 7.4% and 16.4% for estradiol,
8.7% and 18.5% for total testosterone,14.9% and 17.2% for free testosterone, and 4.9% and
4.6% for SHBG. Serum levels of progesterone were compatible with ovulatory cycles,
ranging between 5.3 and 21.5 ng/ml in control subjects and between 4.8 and 20.8 ng/ml in
incident breast cancer cases.

Statistical analyses
Based on their distribution, we applied logarithmic transformations to the data for LH,
FSH, and SHBG, and square-root transformations to total testosterone, free testosterone,
progesterone, and estradiol. We evaluated differences between case and control means by
using the t test for paired data or the Wilcoxon signed test, as appropriate. Pearson partial
correlation coefficients, adjusted for age and case-control status, were computed to examine
the strength of linear associations among the various hormones and between each hormone,
the waist circumference, and the body mass index (BMI).
We used conditional regression models to estimate the relative risks of breast cancer
(reported as odds ratios (ORs) with 95% confidence intervals (CIs)) by tertiles of circulating
sex steroids, which were defined on the basis of the values for all control subjects. We used
likelihood ratio tests to assess linear trends in odds ratios, with increasing exposure level as a
continuous variable.
The effects of additional potential confounders (other than the matching criteria,
which were controlled by study design) were examined by including additional regression
terms into the logistic regression models. Potential confounders included age at recruitment,
BMI, because of its role in the hormone metabolism, education, and years of education.
Furthermore, the better to characterize the period of the ovarian cycle at blood drawing (12),
multivariate models were adjusted for variables related to the timing of the ovarian cycle at
blood drawing, such as time between date of last menses before blood sampling and day of
blood sampling, time between date of menses after blood sampling and date of blood
sampling, circulating FSH and LH levels, and menstrual-cycle length. We used STATA
version 8 for all analyses. All P values were two-sided.

Results
Study participants were all premenopausal, with an age range of 35 to 54 years at
blood collection. Most of the women’s baseline characteristics did not differ by casecontrol
status (Table 1), in particular, for the reproductive variables.
In Table 2 partial correlation coefficients, adjusted for case-control status and age at
blood donation, indicated that serum concentration of estradiol (E2) statistically significantly
correlated with all the other hormones, with correlation coefficients (r) ranging from 0.20
with progesterone to 0.38 with total testosterone. Also, the sex hormone-binding globulin
(SHBG) statistically significantly correlated with most of hormones, with the highest positive
correlation with estradiol (r = 0.32). We also observed that SHBG had significant negative
correlation with BMI (r = -0.25), waist-to-hip ratio (r = -0.21), FSH (r = -0.12), and free
testosterone (r = -0.22). Both total testosterone and free testosterone correlated with estradiol
(free testosterone, r = 0.22) and LH (r = 0.22 and r = 0.11 for total and free testosterone,
respectively). In addition, free testosterone correlated with BMI (r = 0.16). For all study
subjects combined, women who developed breast cancer had statistically significantly higher
mean levels of free testosterone. Specifically, we observed a significant association for
circulating free testosterone (OR for highest versus lowest tertile, 2.43; 95% CI, 1.15 to 5.10;
Ptrend = 0.03; Table 3).
We also observed a positive albeit nonsignificant association for circulating total
testosterone (OR for highest versus lowest tertile, 1.27; 95% CI, 0.62 to 2.61; Ptrend = 0.51).
Further, none of the other sex steroids evaluated showed significant associations with breast
cancer risk in this data set. We examined the effects of various confounders on these relative-
risk estimates by performing adjusted analyses. Relative-risk estimates remained virtually
unchanged after adjustments for BMI and education. We also evaluated potential variation in
the risk estimates for free testosterone by adjusting in different models for progesterone, total
testosterone, estradiol, and SHBG, and by excluding breast cancer cases diagnosed within 2
years after blood collection (n = 35), and all the risk estimates remained very similar (data not
shown).
Discussion
This study was designed to provide information on breast cancer risk in relation to
serum levels of sex hormones that markedly vary over the menstrual cycle. To reduce this
variability, blood samples were collected within a narrow window (days 20 to 24 of the
cycle) when most women were in midluteal phase. Then we adjusted our estimates for
variables related to ovarian-cycle time intervals and restricted the analyses to women with
regular cycles throughout their life.
Our findings are largely in line with those of previous reports, which have found few
consistent associations between sex steroids and breast cancer risk in premenopausal women,
with the exception of the consistent risk elevation associated with testosterone and free
testosterone. Of the prospective studies published to date [4-16], the two largest ones, based
in the European Prospective Investigation into Cancer and Nutrition (EPIC) and Nurses’
Health Study II (NHS2) cohorts, reported for circulating estradiol either no overall
association [4] or only if estradiol levels were measured in the follicular phase of the
menstrual cycle [11]. Our blood samples were collected exclusively in the luteal phase of the
menstrual cycle, and even though we accounted for the exact day within a woman’s
menstrual cycle at which blood was drawn, we did not observe an association between luteal
circulating estradiol and breast cancer risk in our analyses, which is in line with the findings
from the NHS cohorts [11]. For progesterone, previous reports were conflicting, reporting no
[11] or a significant inverse association [4] with breast cancer risk. Similarly, few and mostly
small prospective studies have explored the association between circulating testosterone and
premenopausal breast cancer risk. Both the European Prospective Investigation into Cancer
(EPIC) and Nurses’ Health Study (NHS) cohorts [4,11], the two largest studies to date, report
positive associations, particularly if testosterone was measured in the luteal phase [11]. We,
too, observed a significantly increased risk of breast cancer associated with higher circulating
levels of free testosterone. We did not observe an association between SHBG and breast
cancer risk in our study, which is also consistent with prior evidence [4,8,9,11,12].
Our study has several important strengths, relating to its prospective nature and
carefully timed sample collection, as well as information on important confounders. This
study is unique in that we were able to collect information on a women’s history of
menstrual-cycle irregularities throughout life, which allowed us subsequently to restrict it to
women with very consistent hormone profiles over their entire reproductive period. Our
results support that, in premenopausal women, it is imperative to apply a strict control of the
biologic variability of hormones and/or other biologic variables to test potential associations
with specific outcomes. In our sensitivity analyses, excluding cases diagnosed within 2 years
after blood collection, or in situ breast cancers, did not alter our findings.
Our study is limited by its relatively small sample size (even though it still ranks
among the larger studies published), which precluded more-detailed stratified analyses.
Whether a single blood measurement of sex steroid hormones, which fluctuate during the
menstrual cycle, sufficiently characterizes a premenopausal woman’s long-term hormone
levels is another concern. However, previous studies showed, particularly for androgens,
estrone sulfate, and to a lesser degree for estradiol and progesterone, that a single
measurement can reliably categorize average levels over at least a 3-year period in
premenopausal women [24]. Nonetheless, the necessity to better characterize and measure the
exposure to serum hormones called for a careful assessment of menstrual-cycle variability.
We excluded these women from our dataset, reducing sample size but substantially
improving our ability to detect associations that appear to become apparent only when
biologic variability is controlled for. In our analyses, no mathematical correction was made
for multiple comparisons. However, although such correction would have diminished
significance (for example, after Bonferroni correction, Ptrend < 0.01 would be considered
statistically significant, and none of our P values met this threshold), it does not appear to be
needed, given that we conducted only a few scientifically sensible comparisons.

Conclusions
Our findings suggest that premenopausal circulating testosterone, particularly free
testosterone, is associated with breast cancer risk when ovarian-cycle variability is carefully
controlled for. We also show that other sex steroids do not appear to play a major role in
developing premenopausal breast cancer. As evidence continues to accumulate for a positive
association between premenopausal circulating testosterone and breast cancer risk, potential
new preventive strategies should be designed and tested.